Free tissue transfer for burns reconstruction: A single-site experience.

This study reviews free tissue transfer (FTT) surgery for both acute wound and reconstructive scar management of burn injuries at a UK burns unit over a 10-year period. Thirty eight patients underwent 46 FTTs, or free flaps, as part of their burn injury pathway. For the cohort of patients, there was one flap failure, which occurred for a secondary scar reconstruction. It is noted that FTT was successful for all seven acute or primary interventions. Anterolateral thigh flap was the most frequently performed (57%); followed by parascapular flaps (22%) of which 43% were pre-expanded. A method of pre-expansion for neck contractures and a novel technique of anchoring this flap to the pre-tracheal fascia are described here. This can provide the patient with good neck contouring by using the capsule to hitch the flap into a good position. It is clear that further work is required to study the prevention of hypertrophic scarring that can occur at the interface between flap and adjacent skin, where occurrence rate in this cohort was 17%. It is proposed that FTT now provides a viable solution both to the coverage of complex burn wounds and to the revision of scar contractures. Consensus over an FTT protocol for the primary management of open burn wounds is seen as the logical next step for this surgical intervention.

Physiologically Based Pharmacokinetic Model to Assess the Influence of Blinatumomab-Mediated Cytokine Elevations on Cytochrome P450 Enzyme Activity.

Blinatumomab is a CD19/CD3 bispecific T-cell engager (BiTE®) antibody construct for treatment of leukemia. Transient elevation of cytokines (interleukin (IL)-6, IL-10, interferon-gamma (IFN-γ)) has been observed within the first 48 hours of continuous intravenous blinatumomab infusion. In human hepatocytes, blinatumomab showed no effect on cytochrome P450 (CYP450) activities, whereas a cytokine cocktail showed suppression of CYP3A4, CYP1A2, and CYP2C9 activities. We developed a physiologically based pharmacokinetic (PBPK) model to evaluate the effect of transient elevation of cytokines, particularly IL-6, on CYP450 suppression. The predicted suppression of hepatic CYP450 activities was <30%, and IL-6-mediated changes in exposure to sensitive substrates of CYP3A4, CYP1A2, and CYP2C9 were

On the anticipation of the human dose in first-in-man trials from preclinical and prior clinical information in early drug development.

The drug development process is divided into phases with decisions required on compound selection and promotion to each subsequent development phase. In preclinical drug development the main objective is to bring the compound into human trials and there is an inability of many preclinical information packages to predict clinical responses. Since clinical responses are functions of the dose, the human dose anticipation should be a key deliverable of any preclinical package of drug candidate. The human dose should be anticipated by integration of information from multiple sources, in vitro and in vivo, non-human and human, using a variety of methodologies and approaches. Prediction of human safe and active dose relies on the availability of validated animal models for effect. Although there are many exceptions to the rule, the paper defines a four-step approach for the anticipation of human dose for first-in-man trials: 1, characterization of non-human exposure-response relationships; 2, correction for interspecies differences; 3, diagnosing compound absorption, distribution, metabolism and excretion (ADME) properties and prediction of human pharmacokinetics; and 4, prediction of human dose-responses and dose selection for phase I protocols.

Pharmacokinetics and haemodynamics of ketamine in intensive care patients with brain or spinal cord injury.

BACKGROUND: Ketamine is used as an anaesthetic agent for short surgical procedures, and as a sedative and analgesic in intensive care patients. Intensive care patients with brain or spinal cord injury may have physiological changes that could alter the pharmacokinetics of ketamine. The pharmacokinetics of ketamine have been studied in healthy volunteers and in patients undergoing different types of surgery, but no data are available in intensive care patients. METHODS: We determined the pharmacokinetics of ketamine and its active metabolites, norketamine and dehydronorketamine, in 12 intensive care patients with brain or spinal cord injury. The effect of ketamine on haemodynamic variables was also investigated. RESULTS: The total clearance of ketamine, mean (SD), was 36.0 (13.3) ml min(-1) kg(-1), the volume of distribution (Vbeta) was 16.0 (8.6) litre kg(-1), and the elimination half-life was 4.9 (1.6) h. Ketamine did not alter any haemodynamic variables in the patients studied. CONCLUSIONS: Pharmacokinetic variables of ketamine in intensive care patients are greater than in healthy volunteers and in surgical patients. The increase in the volume of distribution is greater than the increase in clearance, resulting in a longer estimated half-life of ketamine in this patient group.

Seroprevalence of West Nile, Rift Valley, and sandfly arboviruses in Hashimiah, Jordan.

We conducted a serosurvey among patients of a health center in Hashimiah, a Jordanian town of 30,000 inhabitants located near a wastewater treatment plant and its effluent channel. Serum samples from 261 patients >/=5 years of age were assessed for immunoglobulin G (IgG) and IgM antibodies against West Nile, sandfly Sicilian, sandfly Naples, and Rift Valley viruses; the seroprevalence of IgG antibodies was 8%, 47%, 30%, and 0%, respectively. Female participants were more likely to have been infected than male. Persons living within 2 km of the treatment plant were more likely to have been infected with West Nile (p=0.016) and sandfly Sicilian (p=0.010) viruses. Raising domestic animals within the house was a risk factor for sandfly Sicilian (p=0.003) but not for sandfly Naples virus (p=0.148). All serum samples were negative for IgM antibodies against the tested viruses. Our study is the first documentation of West Nile and sandfly viruses in Jordan and calls attention to the possible health hazards of living close to wastewater treatment plants and their effluent channels.

Neuroectodermal differentiation in Ewing's sarcoma family of tumors does not predict tumor behavior.

The observation that neuroectodermal differentiation imparts a worse prognosis to the Ewing family of tumors has been suggested by some studies and refuted by others. To assess whether the diagnosis of Ewing's sarcoma versus peripheral primitive neuroectodermal tumor (PNET) affects prognosis, we analyzed tumors from 63 analogously treated pediatric and young adult patients from the National Cancer Institute and St Jude Children's Research Hospital and retrospectively compared the results with clinical outcomes. The tumors were assessed using standard light microscopy and immunohistochemical stains for neuron-specific enolase, CD57, S100 protein, neurofilament protein, and synaptophysin with or without antigen retrieval. Ultrastructural evaluation was also performed in 39 tumors. Classification was performed using Kiel criteria as well as a modified classification. Kaplan-Meier analyses, with Mantel-Haenzel evaluation of the significance of the differences, were performed separately for localized or metastatic tumors. Using the Kiel classification on a subset of 60 cases, 39 tumors qualified as PNET and 21 as Ewing's sarcoma. Using the modified classification on a subset of 61 cases, 14 were classified as PNET, 21 as atypical Ewing's sarcoma, and 26 as Ewing's sarcoma. The addition of electron microscopy to the diagnostic armamentarium significantly increased the likelihood of identifying PNET. No significant differences in event-free or overall survival were seen using either the modified or Kiel classification, regardless of the ancillary diagnostic techniques employed. In this exploratory analysis, neuroectodermal differentiation did not play a role in clinical outcome. Confirmation of this finding will require a larger, separate study of similarly treated patients, and it may not apply to older patients.

Melanoma-associated antigen recognized by T cells (MART-1): the advent of a preferred immunocytochemical antibody for the diagnosis of metastatic malignant melanoma with fine-needle aspiration.

BACKGROUND: HMB-45, an antibody directed against a premelanosome glycoprotein, has thus far been considered the most specific antibody for the immunocytochemical substantiation of the diagnosis of malignant melanoma (MM). A recently described antigen, MART-1, is a transmembrane protein that is present in normal melanocytes and widely expressed in MM. Antibodies to MART-1 have recently become commercially available. Both HMB-45 and MART-1 form the basis of ongoing immunotherapy protocols at the National Institutes of Health/National Cancer Institute. METHODS: The authors evaluated 207 lesions from 160 patients with metastatic MM procured via fine-needle aspiration (FNA) for expression of MART-1 (clone M2-7C10) and HMB-45 prior to commencement of immunotherapy. FNAs were performed on subcutaneous soft tissue masses (190 lesions), lung (8 lesions), liver (5 lesions), pancreas (3 lesions), and brain (1 lesion). To test the specificity of the monoclonal antibody directed against MART-1, the authors evaluated its reactivity in normal tissues as well as in various nonpigmented neoplasms that are often included in the differential diagnosis of MM. RESULTS: Of all lesions tested, 13 (6%) were negative for both MART-1 and HMB-45. Of all patients tested, 20% had 1 or more lesions that were non-immunoreactive with HMB-45, whereas only 10% had 1 or more lesions that were nonimmunoreactive for MART-1. Eight percent of the lesions tested were negative for MART-1 only, whereas 16% of lesions tested were negative for HMB-45 only. In 35% of the lesions, MART-1 stained more cells than HMB-45. In 13%, MART-1 stained fewer cells than HMB-45, and in 52% both antibodies stained an equivalent number of cells. All samples of normal tissue were negative for staining with MART-1, as were the nonpigmented lesions tested. Melanocytes in normal skin samples stained positively for MART-1. CONCLUSIONS: The MART-1 antibody is a superior immunohistochemical marker for the diagnosis of MM. It has the potential to become the preferred antibody over HMB-45 for the diagnosis of metastatic MM in FNA material, as MART-1 stains a higher percentage of lesions in a higher percentage of patients than does HMB-45.

Utility of the antibodies CA 19-9, HBME-1, and thrombomodulin in the diagnosis of malignant mesothelioma and adenocarcinoma in cytology.

BACKGROUND: The distinction between malignant mesothelioma (MM) and adenocarcinoma (ACA) in cytologic specimens frequently is difficult, often requiring immunocytochemistry to support the diagnosis. Recent reports have proposed the utilization of antibodies to mesothelial cell clone HBME-1 and thrombomodulin (TM), because they are immunoreactive in MM and less commonly reactive in ACA. Immunoreactivity for the monoclonal antibody CA 19-9 has been observed in many ACAs and reportedly is absent in MM. METHODS: In this study, immunostaining was performed on formalin fixed, paraffin embedded cell blocks from effusions or fine-needle aspirations using the avidin-biotin-peroxidase method. Thirty-eight MMs and 49 ACAs were tested using antibodies to CA 19-9, HBME-1, and TM. RESULTS: Anti-CA 19-9 stained only 1 of the 37 cases of MM tested (3%), but stained 24 of the 49 cases of ACA (49%). Anti-HBME-1 stained 34 of 38 cases of MM (89%), and 28 of 43 cases of ACA tested (65%). Anti-TM stained 24 of 36 cases of MM (67%), and 21 of 40 cases of ACA tested (53%). CONCLUSIONS: CA 19-9 has utility as part of an immunocytochemical panel for distinguishing ACA from MM, because a positive staining reaction would make the diagnosis of MM unlikely. Although HBME-1 and TM can identify MM positively, each frequently is detected in ACA, thus limiting the utility of these antibodies in cytologic specimens.

Use of recombinant poxviruses to stimulate anti-melanoma T cell reactivity.

BACKGROUND: Dendritic cells (DC) are potent professional antigen-presenting cells that can activate naive T lymphocytes and initiate cellular immune responses. As adjuvants, DC may be useful for enhancing immunogenicity and mediating tumor regression. Endogenous expression of antigen by DC could offer the potential advantage of allowing prolonged constitutive presentation of endogenously processed epitopes and exploitation of multiple restriction elements for the presentation of the same antigen. METHODS: DC were prepared from the peripheral blood of HLA A*0201 patients with metastatic melanoma in the presence of IL-4 (1000 IU/mL) and GMCSF (1000 IU/mL). Recombinant vaccinia and fowlpox viruses encoding the hMART-1 gene were constructed and used to infect DC. The efficiency of infection and expression of the MART-1 antigen were assessed by immunohistochemistry and intracellular FACS analyses. Cytotoxic lymphocytes (CTL) were generated by the stimulation of CD8+ T cells, with DC expressing the recombinant gene. Reactivity of the CTL was determined at weeks 1 and 2 by the amount of IFN-gamma released. RESULTS: DC were infected with recombinant poxviruses and demonstrated specific melanoma antigen expression by immunohistochemistry, immunofluorescence, and intracellular FACS analysis. The expression by DC of MART-1 MAA after viral infection was sufficient to generate CD8+ T lymphocytes that recognized naturally processed epitopes on tumor cells in 10 of 11 patients. CONCLUSIONS: Human DC are receptive to infection by recombinant poxviruses encoding MAA genes and are capable of efficiently processing and presenting these MAA to cytotoxic T cells. The potential advantage of this approach is the ability to present specific antigen independent of the identification of the epitope or the MHC restriction element. This strategy may be useful for the identification of relevant epitopes for a diverse number of HLA alleles and for active immunization in patients.

Heterogeneous expression of melanoma-associated antigens and HLA-A2 in metastatic melanoma in vivo.

MART-1/MelanA and Pmel17/gp100 are melanoma-associated antigens (MAAs) that can be recognized by tumor-infiltrating lymphocytes (TILs) capable of mediating successful adoptive therapy in vivo. Analysis of melanoma cell lines in vitro has demonstrated that heterogeneous antigen expression in the context of class I MHC is a significant co-factor in determining the recognition of melanoma targets by cytotoxic lymphocytes (CTLs). In this study, 217 specimens from 103 patients with metastatic melanoma were examined for the expression of MART-1/MelanA (monoclonal antibody [MAb] M27C10) and Pmel17/gp100 (HMB45 MAb) by immuno-histochemistry. Marked heterogeneity in the expression of both MAAs was confirmed by analysis of the percentage of positively staining tumor cells or the average intensity of tumor staining. We also noted heterogeneity of expression among multiple lesions taken from different anatomic sites within a patient. A dissociation was noted in the detection of MART-1 and gp100 in some lesions, with gp100 being undetectable in 24% of the lesions and MART-1 being undetectable in 11%. In several cases, loss of one MAA was not associated with loss of the other MAA, suggesting that MART-1 can represent a useful additional marker for the diagnosis of melanoma in gp100 (HMB45)-negative lesions. Of the 217 specimens, 155 were obtained from HLA-A*0201 patients, of which 6% were negative for HLA-A2, 8% were negative for MART-1/MelanA and 21% were negative for Pmel17/gp100. The potential significance of our findings is illustrated by a case study in which a patient with melanoma experienced rapid tumor progression in association with loss of either MAA or HLA expression in several lesions.

Comparative analysis of the in vivo expression of tyrosinase, MART-1/Melan-A, and gp100 in metastatic melanoma lesions: implications for immunotherapy.

A variety of human melanoma-associated antigens (MAA) have been identified that can be recognized by T lymphocytes in a major histocompatibility complex-restricted fashion. Among them, tyrosinase, MART-1/Melan- A, and gp100 are derived from nonmutated melanocyte lineage-specific antigens (Ag). These Ag can be recognized by CD8+ and, in the case of tyrosinase, CD4+ T cells. The in situ expression of these MAA may be a significant cofactor in determining the recognition of melanoma targets by Ag-specific T cells. In this study, we examined the patterns of expression of these MAA using immunohistochemical methods on 30 metastatic tumor deposits derived from 25 patients. MAA expression was heterogeneous among the 30 specimens and also within individual lesions. Of note, 23% of the samples examined failed to express the gp100 protein, and 17% of samples had no detectable expression of MART-1. In contrast, all lesions demonstrated some degree of tyrosinase expression even in cases where both gp100 and MART-1 were not detectable. In addition, 60% of samples (18 of 30) showed strong positivity for tyrosinase (> 75% of cells staining) compared with 40% for gp100 and 36% for MART-1. Currently, a number of experimental immunotherapies for melanoma are directed against the MAA tyrosinase, MART-1, and gp100. Although threshold levels of Ag required for T-cell recognition have not yet been defined, tumor-associated Ag expressed in high density, such as tyrosinase, may be better targets for future immunotherapy trials.

Calcium ionophore-treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated dendritic cells.

Human peripheral blood contains a small subpopulation of immature dendritic cells (iDC) distinguished from circulating monocytes by their low expression of CD14. We utilized leukapheresis and countercurrent centrifugal elutriation to obtain myeloid origin mononuclear cell (MOMC) fractions of monocytes and iDC for study. These subpopulations were ultrastructurally and immunophenotypically similar before culture. After a 20- to 96-h culture either alone, with recombinant human granulocyte-monocyte CSF, or with endotoxin, greater up-regulation of costimulatory molecule expression was observed among iDC than among monocytes, and only iDC expressed the activation molecule CD83. Treatment with rhIL-4 caused many MOMC to develop morphologic properties of dendritic cells within 96 h, but costimulatory molecule up-regulation and CD14 down-regulation were heterogeneous, and CD83 expression was infrequent. In contrast, calcium ionophore (CI) treatment induced rapid and consistent effects in MOMC from both healthy volunteers and cancer patients, including down-regulated CD14 expression, acquisition of dendritic cell morphologic properties, up-regulated MHC and costimulatory molecule expression, and de novo CD83 expression. Many such effects occurred within 20 h of treatment. CI treatment activated purified CD14+ monocytes and also enhanced the spontaneous activation of purified CD14-/dim iDC in culture. Unfractionated MOMC, purified monocytes, and purified iDC displayed equivalently enhanced T cell-sensitizing efficiency following CI treatment. CD4+ T cell sensitization to keyhole limpet hemocyanin and CD8+ T cell sensitization to MART-1 melanoma-associated peptide were achieved in a single culture stimulation. Therefore, circulating monocytes and iDC can be induced by CI to manifest properties of activated DC, providing large numbers of efficient, nontransformed autologous APC for T cell sensitization strategies.

Immunocytochemical detection of MART-1 in fresh and paraffin-embedded malignant melanomas.

MART-1 is a transmembrane protein that appears to be melanocyte specific. This study evaluated the use of a new monoclonal antibody, directed against MART-1, in fresh and paraffin-embedded specimens. Twenty-three paraffin-embedded tumors were evaluated for MART-1 immunoreactivity both before and after microwave-based antigen retrieval. After this comparison, 53 fine-needle aspirates from 43 patients with malignant melanoma were assessed for MART-1 immunoreactivity. Immunocytochemistry was performed on both cytospins and paraffin-embedded tissues that were pretreated with antigen retrieval. Seventeen (74%) of 23 tumors evaluated for immunoreactivity before and after antigen retrieval showed a significant increase in both staining intensity and the number of cells stained. When cytospins and antigen-retrieved cell blocks from 53 fine-needle aspirates were compared. 38 (72%) showed good correlation. In 13 (25%) of 53 tumors, MART-1 immunoreactivity was more intense in the cytospins, although the differences were marked in only two cases: Microwave-based antigen retrieval renders paraffin-embedded tissues nearly as sensitive as fresh material for use in the immunocytochemical detection of MART-1. This technique will allow the evaluation of MART-1 immunoreactivity in archival malignant melanomas.

Tumor-infiltrating lymphocytes from human ovarian cancer patients recognize autologous tumor in an MHC class II-restricted fashion.

PURPOSE: To search for tumor-specific in vitro reactivity by lymphocytes derived from patients with ovarian carcinoma. METHODS: Tumor-infiltrating lymphocytes (TIL) were derived from primary or metastatic solid tumors and tumor-associated lymphocytes (TAL) were derived from ascites from 13 patients with ovarian cancer. TIL or TAL were cultured for approximately 30 to 60 days and studied for phenotype, cytotoxicity, and cytokine secretion in response to autologous tumor stimulation. RESULTS: Twenty-nine bulk TIL or TAL cultures were successfully established from 10 patients using various culture conditions. Thirteen cultures were predominantly CD4+ and 16 were mainly CD8+. In contrast to reports by others, none of the cultures tested were specifically lyric for autologous tumor. Five predominantly CD4+ bulk TIL (from four patients) preferentially secreted tumor necrosis factor-alpha and granulocyte macrophage-colony stimulating factor when stimulated with autologous tumor and not when stimulated by autologous Epstein Barr virus-B cells, fibroblasts, peripheral blood mononuclear cells, or allogeneic HLA matched or mismatched stimulators. This cytokine secretion was found to be MHC class-II restricted in three patients because it was inhibited by the anti-MHC class-II antibody IVA12 and the HLA-DR specific antibody L243. CONCLUSION: We believe these data are the first to suggest that tumor reactive CD4+ lymphocytes exist in some ovarian cancer patients. This finding may be useful in the development of novel immunotherapies for these patients.

Ifosfamide and etoposide plus vincristine, doxorubicin, and cyclophosphamide for newly diagnosed Ewing's sarcoma family of tumors.

BACKGROUND: This study was conducted to determine the feasibility of, and improve outcome by, incorporating ifosfamide and etoposide (IE) into the therapy of newly diagnosed patients with Ewing's sarcoma family of tumors of bone and soft tissue. METHODS: Fifty-four newly diagnosed patients received 7 cycles of vincristine, doxorubicin, and cyclophosphamide (VAdriaC) and 11 cycles of IE. Radiation therapy after the fifth chemotherapy cycle was the primary approach to local control. RESULTS: Actuarial 5-year event-free survival (EFS) and overall survival rates were 42% and 45%, respectively, with a median duration of potential follow-up of 6.8 years. EFS was significantly better for patients with localized tumors than for those with metastatic lesions (64% v. 13%, P < 0.0001). Actuarial local progression-free survival at 5 years was 74%, and did not correlate with primary tumor size or site, histologic subtype, or the presence of metastases. Febrile neutropenia developed after 49% of cycles, and clinical or sub-clinical cardiac dysfunction was common (7% and 40% respectively). There were four toxic deaths and one case of secondary myelodysplastic syndrome. CONCLUSIONS: Despite substantial toxicity, the integration of IE into the front-line, VAdriaC-based therapy of patients with Ewing's sarcoma family of tumors is feasible and appeared to significantly improve the outcome for patients with high risk localized tumors, but had no impact on the poor prognosis of patients with metastatic tumors. Local control can be achieved in the vast majority of patients using radiotherapy exclusively, even among patients with bulky, central axis tumors. Longer follow-up is needed to evaluate the late effects of this intensive therapy.

Application of molecular techniques to the diagnosis of microsporidial infection.

Microsporidia are now recognized as important pathogens of AIDS patients; the ability of these parasites to cause disease in immunocompetent persons is still being elucidated. Improved diagnostic tests for microsporidial infection are continually being sought for establishing diagnosis in order to avoid laborious electron microscopy studies that require invasively acquired biopsy specimens. Modified trichrome or chemofluorescent stains are useful for detecting microsporidia in bodily fluids and stool specimens, but they do not allow for speciation of microsporidia. Polymerase chain reaction with specific primers will allow the detection and speciation of microsporidia in biopsy tissue, bodily fluids, and stool specimens.

Oncocytic nodular hidradenoma.

Oncocytic neoplasms are most commonly of salivary, thyroid, parathyroid, and renal origin. Ocular adnexal tumors with oncocytosis have been reported. We report an unusual example of a skin adnexal tumor from the back. A tumor of uncertain duration was excised from a 54-year-old man. Light microscopy of hematoxylin and eosin-stained sections showed a large, fairly well-defined cystic nodular hidradenoma with areas infiltrated by numerous closely arranged, large, uniform, oval and polygonal cells with abundant intensely eosinophilic cytoplasm and small central, dark, round nuclei. No significant cellular atypia or mitotic figures were observed. The cytoplasm of these cells showed markedly positive immunostaining with monoclonal antimitochondrial antibodies. Electron microscopy demonstrated cytoplasm packed with mitochondria. Pure oncocytic tumors usually follow a benign clinical course. The focal presence of oncocytes in an otherwise histologically recognizable tumor apparently does not affect the prognosis, which in this case is the favorable outcome expected for a nodular hidradenoma.

Cytologic effects of photodynamic therapy in body fluids.

Photodynamic therapy (PDT) has been used in phase I clinical trials at the National Institutes of Health for the treatment of malignancies disseminated within the peritoneal and pleural cavities. Review of records revealed 18 patients who were treated with PDT between April 1988-June 1993. Sixty-five pleural and peritoneal fluids, 22 pre- and 43 post-PDT, were available for evaluation. Mesothelial cell changes seen post-PDT included: increased nuclear-to-cytoplasmic ratios in 7/18 (39%), cytomegaly in 9/18 (50%), and multinucleation in 12/18 (67%), with Touton-like giant cells in 3/18 (17%). Additional changes noted post-PDT comprised histiocytic aggregates in 9/18 patients (50%), with granuloma-like clusters in 3/18 (17%), acute and chronic inflammation in 13/18 (72%), and eosinophilia in 8/18 (44%). Residual tumor was present in 7/18 (39%) patients post-PDT. In 2 patients with malignant mesothelioma, benign mesothelial cells with cytologic changes post-PDT were difficult to distinguish from malignant cells. Mesothelial cell changes following PDT, specifically increased nuclear-to-cytoplasmic ratios and cytomegaly, should be recognized to avert false-positive diagnoses of tumor. In patients with malignant mesothelioma, and less commonly with adenocarcinoma, benign mesothelial cells with changes secondary to PDT may be difficult to distinguish from tumor cells.